The present invention relates generally to mitochondria protecting agents for treating diseases in which mitochondrial dysfunction leads to tissue degeneration and, more specifically, to compounds, compositions and methods for treating such diseases.
Mitochondria are the subcellular organelles that manufacture essential adenosine triphosphate (ATP) by oxidative phosphorylation. A number of degenerative diseases may be caused by or associated with either direct or indirect alterations in mitochondrial function. These include Alzheimer""s Disease, diabetes mellitus, Parkinson""s Disease, neuronal and cardiac ischemia, Huntington""s disease and other related polyglutamine diseases (spinalbulbar muscular atrophy, Machado-Joseph disease (SCA-3), dentatorubro-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxias 1, 2 and 6), dystonia. Leber""s hereditary optic neuropathy, schizophrenia, and myodegenerative disorders such as xe2x80x9cmitochondrial encephalopathy, lactic acidosis, and strokexe2x80x9d (MELAS), and xe2x80x9cmyoclonic epilepsy ragged red fiber syndromexe2x80x9d (MERRF).
Defective mitochondrial activity, including but not limited to failure at any step of the elaborate multi-complex mitochondrial assembly, known as the electron transport chain (ETC), may result in 1) decreases in ATP production, 2) increases in the generation of highly reactive free radicals (e.g., superoxide, peroxynitrite and hydroxyl radicals, and hydrogen peroxide). 3) disturbances in intracellular calcium homeostasis and/or 4) release of apoptosis inducing factors such as, e.g., cytochrome c. Because of these biochemical changes, mitochondrial dysfunction has the potential to cause widespread damage to cells and tissues For example, oxygen free radical induced lipid peroxidation is a well established pathogenetic mechanism in central nervous system (CNS) injury such as that found in a number of degenerative diseases, and in ischemia (i.e., stroke).
Mitochondrial dysfunction also is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1277-87, 1995). Altered mitochondrial physiology may be among the earliest events in apoptosis (Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995. In several cell types, including neurons, reduction in the mitochondrial membrane potential (xcex94"psgr"m), a sign of mitochondrial dysfunction, precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, mitochondrial, but not nuclear, enriched fractions are capable of inducing nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratory activity leading to altered cellular metabolic states may occur in mitochondria associated diseases and may further induce pathogenetic events via apoptotic mechanisms. For example, altered mitochondrial activity may lead to undesirable elevated levels of intracellular reactive oxygen species (ROS) and subsequent intracellular damage or cell death.
Stressed (e.g., stressors included free radicals, high intracellular calcium. loss of ATP, among others) mitochondria may release preformed soluble factors that can initiate apoptosis through an interaction with novel apoptosomes (Marchetti et al., Cancer Res. 56:2033-38, 1996; Li et al., Cell 91:479-89, 1997). Release of preformed soluble factors by stressed mitochondria, like cytochrome c, may occur as a consequence of a number events. In some cases, release of apoptotic molecules (apoptoqens) occurs when mitochondria undergo a sudden change in permeability to cytosolic solutes under 1.5 KDa. This process has been termed permeability xe2x80x9ctransitionxe2x80x9d. In other cases, the permeability may be more subtle and perhaps more localized to restricted regions of a mitochondrion. In still other cases, overt permeability transition may not occur but apoptogens can still be released as a consequence of mitochondrial abnormalities. Thus, changes in mitochondrial physiology may be important mediators of apoptosis. To the extent that apoptotic cell death is a prominent feature of degenerative diseases, mitochondrial dysfunction may be a critical factor in disease progression.
Diabetes mellitus is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing diabetes mellitus is reportedly maternally inherited, suggesting a mitochondrial genetic involvement (Alcolado et al., Br. Med. J. 302:1178-1180, 1991; Reny, International J. Epidem. 23:886-890, 1994). Diabetes is a heterogeneous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.
At the cellular level the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function for example impaired insulin secretion and responsivity decreased ATP synthesis and increased levels of reactive oxygen species. Studies have shown that diabetes mellitus may be preceded by or associated with certain related disorders. For example, it is estimated that tort million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. A small percentage (5-10%) of IGT individuals progress to insulin deficient non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). These forms of diabetes mellitus. NIDDM and IDDM, are associated with decreased release of insulin by pancreatic beta cells and/or a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies, blindness and deafness.
Due to the strong genetic component of diabetes mellitus, the nuclear genome has been the main focus of the search for causative genetic mutations. However, despite intense effort, nuclear genes that segregate with diabetes mellitus are known only for rare mutations in the insulin gene, the insulin receptor gene, the adenosine deaminase gene and the glucokinase gene. Accordingly, mitochondrial defects, which may include but need not be limited to defects related to the discrete non-nuclear mitochondrial genome that resides in mitochondrial DNA, may contribute significantly to the pathogenesis of diabetes mellitus.
Parkinson""s disease (PD) is a progressive, mitochondria associated neurodegenerative disorder characterized by the loss and/or atrophy of dopamine-containing neurons in the pars compacta of the substantia nigra of the brain. Like Alzheimer""s Disease (AD), PD also afflicts the elderly. It is characterized by bradykinesia (slow movement), rigidity and a resting tremor. Although L-Dopa treatment reduces tremors in most patients for a while, ultimately the tremors become more and more uncontrollable, making it difficult or impossible for patients to even feed themselves or meet their own basic hygiene needs.
It has been shown that the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonism in animals and man, at least in part through its effects on mitochondria. MPTP is converted to its active metabolite, MPPxe2x88x92, in dopamine neurons; it then becomes concentrated in the mitochondria. The MPP then selectively inhibits the mitochondrial enzyme NADH:ubiquinone oxidoreductase (xe2x80x9cComplex Ixe2x80x9d), leading to the increased production of free radicals, reduced production of adenosine triphosphate and. ultimately the death of affected dopamine neurons.
Mitochondrial Complex I is composed of 40-50 subunits, most are encoded by the nuclear genome and seven by the mitochondrial genome. Since parkinsonism may be induced by exposure to mitochondrial toxins that affect Complex I activity, it appears likely that defects in Complex I proteins may contribute to the pathogenesis of PD by causing a similar biochemical deficiency in Complex I activity. Indeed, defects in mitochondrial Complex I activity have been reported in the blood and brain of PD patients (Parker et al., Am. J. Neurol. 26:719-723, 1989).
Similar theories have been advanced for analogous relationships between mitochondrial defects and other neurological diseases, including Alzheimer""s disease (AD), Leber""s hereditary optic neuropathy, schizophrenia, xe2x80x9cmitochondrial encephalopathy, lactic acidosis and strokexe2x80x9d (MELAS), and xe2x80x9cmyoclonic epilepsy ragged red fiber syndromexe2x80x9d (MERRF).
For example, AD is a progressive neurodegenerative disorder that is characterized by loss and/or atrophy of neurons in discrete regions of the brain, and that is accompanied by extracellular deposits of xcex2-amyloid and the intracellular accumulation of neurofibrillary tangles. It is a uniquely human disease, affecting over 13 million people worldwide. It is also a uniquely tragic disease. Many individuals who have lived normal, productive lives are slowly stricken with AD as they grow older, and the disease gradually robs them of their memory and other mental faculties. Eventually, they cease to recognize family and loved ones, and they often require continuous care until their eventual death.
There is evidence that defects in oxidative phosphorylation within the mitochondria are at least a partial cause of sporadic AD. The enzyme cytochrome c oxidase (COX), which makes up part of the mitochondrial electron transport chain (ETC), is present in normal amounts in AD patients; however, the catalytic activity of this enzyme in AD patients and in the brains of AD patients at autopsy has been found to be abnormally low (Parker et al., Neurology 44:1086-1090, 1994). This suggests that the COX in AD patients is defective, leading to decreased catalytic activity that in some fashion causes or contributes to the symptoms that are characteristic of AD.
One hallmark pathology of AD is the death of selected neuronal populations in discrete regions of the brain. Cell death in AD is presumed to be apoptotic because signs of cell death are observed whereas indicators of active gliosis and necrosis are not (Smale et al., Exp. Neiurolog. 133:225-230, 1995; Cotman et al., Molec. Neurobiol. 10:19-45, 1995). The consequences of cell death in AD, neuronal and synaptic loss, are closely associated with the clinical diagnosis of AD and are highly correlated with the degree of dementia in AD (DeKosky et al., Ann. Neurology 27:457-464, 1990).
Indeed. focal defects in energy metabolism in the mitochondria, with accompanying increases in oxidative stress, may be associated with AD. It is well-established that energy metabolism is impaired in AD brain (Palmer et al., Brain Res. 645:338-42, 1994: Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275, 1989: Balazs et al., Neurochem. Res. 19:1131-37, 1994; Mecocci et al., Ann Neurol, 36:747-751, 1994; Gsell et al., J. Neurochem. 64:1216-23, 1995). For example, regionally specific deficits in energy metabolism in AD brains have been reported in a number of positron emission tomography studies (Kuhl, et al., J. Cereb. Blood Flow Metab. 7:S406, 1987; Grady, et al., J. Clin. Exp. Neuropsychol. 10:576-96, 1988; Haxby et al., Arch Neurol. 47:753-60, 1990; Azari et al., J. Cereb. Blood Flow Metab. 13:438-47, 1993). Metabolic defects in the temporoparietal neocortex of AD patients apparently presage cognitive decline by several years. Skin fibroblasts from AD patients display decreased glucose utilization and increased oxidation of glucose, leading to the formation of glycosylation end products (Yan et al., Proc. Nat. Acad. Sci. USA 91:7787-91, 1994). Cortical tissue from postmortem AD brain shows decreased activity of the mitochondrial enzymes pyruvate dehydrogenase (Sheu et al., Ann. Neurol 17:444-49, 1985) and xcex1-ketoglutarate dehydrogenase (Mastrogiacomo et al., J. Neurochem. 6:2007-14, 1994), which are both key enzymes in energy metabolism. Functional magnetic resonance spectroscopy studies have shown increased levels of inorganic phosphate relative to phosphocreatine in AD brain, suggesting an accumulation of precursors that arises from decreased ATP production by mitochondria (Pettegrew et al., Neurobiol Of Aging 15:117-32, 1994; Pettigrew et al., Neurobiol. Of Aging 16:973-75, 1995).
Signs of oxidative injury also are prominent features of AD pathology, and reactive oxygen species (ROS) are critical mediators of neuronal degeneration. Indeed, studies at autopsy show that markers of protein, DNA and lipid peroxidation are increased in AD brain probably as a result of increased ROS production secondary to mitochondrial dysfunction (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275-82, 1989; Balazs et al., Arch. Neurol. 4:864, 1994; Mecocci et al., Ann. Neurol. 36:747-51, 1994; Smith et al., Proc. Nat. Acad Sci. USA 88:10540-43, 1991). In hippocampal tissue from AD but not from controls, carbonyl formation indicative of protein oxidation is increased in neuronal cytoplasm, and nuclei of neurons and glia (Smith et al., Nature 382:120-21, 1996). Neurofibrillary tangles also appear to be prominent sites of protein oxidation (Schweers et al., Proc. Nat. Acad. Sci. USA 92:8463, 1995: Blass et al., Arch. Neurol. 4:864, 1990). Under stressed and non-stressed conditions incubation of cortical tissue from AD brains taken at autopsy demonstrate increased free radical production relative to non-AD controls. In addition, the activities increased of critical antioxidant enzymes, particularly catalase, are reduced in AD (Gsell et al., J. Neurochem. 64:121623, 1995), suggesting that the AD brain is vulnerable to increased ROS production. Thus, oxidative stress may contribute significantly to the pathology of mitochondria associated diseases such as AD, where mitochondrial dysfunction and/or elevated ROS may be present.
Accordingly, there is a need for compounds, compositions and methods that limit or prevent damage to organelles, cells and tissues initiated by various consequences of mitochondrial dysfunction. In particular, because mitochondria are essential organelles for producing metabolic energy, agents that inhibit the production of, and/or protect mitochondria and cells against, ROS and other sources of injury would be especially useful. Such agents would be suitable for the treatment of degenerative diseases, including mitochondria associated diseases. The present invention fulfills these needs and provides other related advantages.
Briefly stated. the present invention is directed to the treatment of mitochondria associated diseases by administration to a warm-blooded animal in need thereof an effective amount of a mitochondria protecting agent having one of the following general structures (I) through (IV): 
wherein X1, X2, X3, X4, Y1, Y2, Y3, Y4, Z1, Z2, Z3, W1, W2, W3, A1, R1, R2, R3 and R4 are as identified in the following detailed description.
The compounds of this invention have activity over a wide range of mitochondria associated diseases, including (but not limited to) Alzheimer""s Disease, diabetes mellitus. Parkinson""s Disease, neuronal and cardiac ischemia. Huntington""s disease and other related polyglutamine diseases (spinalbulbar muscular atrophy, Machado-Joseph disease (SCA-3), dentatorubro-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxias 1, 2 and 6), dystonia Leber""s hereditary optic neuropathy, schizophrenia, and myodegenerative disorders such as xe2x80x9cmitochondrial encephalopathy, lactic acidosis, and strokexe2x80x9d (MELAS), and xe2x80x9cmyoclonic epilepsy ragged red fiber syndromexe2x80x9d (MERRF).
Accordingly, this invention is also directed to method of treating a mitochondria associated disease by administration of an pharmaceutically effective mount of a mitochondria protecting agent to a warm-blooded animal in need thereof as well as to pharmaceutical compositions containing a mitochondria protecting agent of this invention in combination with a pharmaceutically acceptable carrier or diluent.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. To that end, various references are set forth herein which describe in more detail certain aspects of this invention, and are each incorporated by reference in their entirety.
The present invention is generally directed to compounds (also referred herein as xe2x80x9cmitochondria protecting agentsxe2x80x9d) and to pharmaceutical compositions containing the same, as well as to methods useful for treating mitochondria associated diseases. More specifically, the mitochondria protecting agents of this invention have an IC50xe2x89xa650 xcexcm, typicallyxe2x89xa61 xcexcm preferablyxe2x89xa6200 nM, and more preferablyxe2x89xa670 nM in the dichlorofluorescin diacetate (DCFC) assay described herein, and have one of the following structures (I) through (IV): 
including steroisomers and pharmaceutically acceptable salts thereof,
where in structure (I):
X1 is selected from xe2x80x94OH, xe2x80x94ORa and xe2x80x94OCOCH3;
Y1 is selected from xe2x80x94OH, xe2x80x94RaOH, xe2x80x94OCOCH3 and C1-12alkyl;
Z1 is selected from xe2x80x94H, xe2x80x94NHH2, xe2x80x94OH, xe2x80x94NO2, xe2x80x94OCOCH3 and C1-12alkyl; and
each occurrence of Ra is selected from C1-6alkyl;
where in structure (II):
R1 and R2 are independently selected from xe2x80x94H, xe2x80x94C(xe2x95x90O)C1-3alkyl and C1-3alkyl;
X2 is optionally present and selected from xe2x80x94(A2)(A3)xe2x80x94, xe2x80x94(CH2)nxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94NHxe2x80x94;
Y2 and Z2 are independently selected from xe2x80x94H, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NO2, xe2x80x94ORb, xe2x80x94NHCNHNH2, xe2x80x94NHRb and xe2x80x94NRbRc;
A2 and A3 are independently selected from xe2x80x94H and C1-3alkyl;
Rb and Rc are independently C1-4alkyl; and
n is 2-9;
where in structure (III):
the dotted line represents a single or double bond;
A1 is selected from xe2x80x94H and C1-3alkyl;
Y3 is selected from xe2x80x94H, C1-3alkyl and xe2x80x94CORd;
Z3 is selected from xe2x80x94H, C1-3alkyl and xe2x80x94(CH,)mX3Rd;
X3 is selected at each occurrence from xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94NHxe2x80x94;
R3 is selected from xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2CH3 and xe2x80x94Rd;
Rd is selected from a guanidino moiety, a cylcoguanidino moiety, and a non-steroidal anti-inflammatory drug; and
m is 1-4; and
where in structure (IV):
W1, W2 and W3 are independently selected from xe2x80x94H and C1-3alkyl;
X4 is optionally selected from xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94Sxe2x80x94;
Y4 is selected from xe2x80x94H and C1-12alkyl; and
R4 is selected from xe2x80x94H, a guanidino moiety, a cylcoguanidino moiety, and a non-steroidal anti-inflammatory drug, or X4xe2x80x94R4 taken together is selected from xe2x80x94CH2OH, xe2x80x94OC(xe2x95x90O)CH3, and C1-3 alkyl.
As used herein, the following terms have the meanings set forth below:
A xe2x80x9cC1-12 alkylxe2x80x9d is a straight chain or branched, saturated or unsaturated hydrocarbon moiety having from 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl butyl, isobutyl, tert-butyl and the like, pentyl, the pentyl isomers, hexyl and the hexyl isomers and the higher homologues having up to 12 carbon atoms such as, for example, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. In instances where a hydrocarbon having a different number of carbon atoms is recited, such as xe2x80x9cC1-3 alkylxe2x80x9d, xe2x80x9cC1-4 alkylxe2x80x9d, xe2x80x9cC1-6 alkylxe2x80x9d, xe2x80x9cC1-8 alkylxe2x80x9d and the like, a straight chain or branched, saturated or unsaturated hydrocarbon moiety is intended, but having the number of carbon atoms indicated.
A xe2x80x9cguanidino moietyxe2x80x9d and a xe2x80x9ccycloguanidino moietyxe2x80x9d have the following structure (a) and structure (b), respectively: 
wherein R5 and R6 are independently selected from xe2x80x94H and C,alkyl, and q is 0-3.
A xe2x80x9cnon-steroidal antiinflammatory drugxe2x80x9d (NSAID) is a NSAID having a carboxylic moiety. A number of chemical classes of NSAID have been identified. The following text, the entire contents of which are hereby incorporated by reference in the present specification, may be referred to for various NSAID chemical classes: CRC Handbook of Eicosanoids: Prostaglandins, and Related Lipids, Volume II, Drugs Acting Via the Eicosanoids, pages 59-133, CRC Press, Boca Raton, Fla. (1989). The NSAID may be selected, therefore, from a variety of chemical classes including, but not limited to, salicylic acid or its derivatives including acetylsalicylic acid, fenamic acids, such as flufenamic acid, nitlumic acid and mefenamic acid; indoles, such as indomethacin, sulindac and tolmetin; phenylalkanoic acids, such as suprofen, ketorolac, flurbiprofen and ibuprofen; and phenylacetic acids, such as dictofenac. Further examples of NSAID include loxoprofen, pirprofen, naproxen, benoxaprofen, aceloferac, fleclozic acid, bromfenac, alcofenac, diflunisal, tolfenamic acid. clidanac, fenclorac, carprofen, fenbufen, amfenac, ketoprofen, orpanoxin, pranoprofen, indoprofen, fenoprofen, meclofenamate, isofezolac, etodolic acid, efenamic acid, fenclofenac, zomopirac, zaltoprofen and other NSAID compounds. The preferred compounds are those wherein the NSAID is selected from the ester or amide derivatives of salicylic acid or its derivatives including acetylsalicylic acid, naproxen, ibuprofen or acetylsalicic acid.
In one aspect, the mitochondria protecting agents of this invention have the following structure (I): 
including steroisomers and pharmaceutically acceptable salts thereof, wherein
X1 is selected from xe2x80x94OH, xe2x80x94ORa and xe2x80x94OCOCH3;
Y1 is selected from xe2x80x94OH, xe2x80x94RaOH, xe2x80x94OCOCH3 and C1-12alkyl;
Z1 is selected from xe2x80x94H, xe2x80x94NH2, xe2x80x94OH, xe2x80x94NO2, xe2x80x94OCOCH3 and C1-12alkyl; and
each occurrence of Ra is selected from C1-6alkyl.
For purpose of clarity, position of Z1 will be referenced by the following numbering: 
In one embodiment of structure (I), X1 is xe2x80x94OH, Z1 is xe2x80x94NH2, and the compounds of this invention have the following structure (I-1): 
Thus, location of the Z1 substituent in structure (I-1) is referred to herein as a xe2x80x9c1xe2x80x94NH2xe2x80x9d.
In one embodiment of structure (I-1), Y1 is C1-12alkyl, such as the following structures (I-2) or (I-3): 
In another embodiment of structure (I), X1 and Z1 are xe2x80x94OH, and the compounds of this invention have the following structure (I-4): 
In one embodiment of structure (I-4), Y1 is C1-12alkyl, such as the following structures (I-5) or (I-6); 
In still another embodiment of structure (I), X1 and Z1 are xe2x80x94OCOCH3, and the compounds of this invention have the following structure (I-7): 
In one embodiment of structure (I-7), Y1 is C1-12alkyl, such as the following structure (I-8): 
In yet another embodiment of structure (I), X1 and Y1 are xe2x80x94OH, and the compounds of this invention have the following structure (I-9): 
In one embodiment of structure (I-9), Z1 is C1-12alkyl, such as the following structure (I-10): 
Representative compounds of structure (I) are set forth in the following Table 1.
The compounds of structure (I) may be made by know organic reaction techniques, including those set forth in Example 6 below. For example. as depicted below under reaction xe2x80x9caxe2x80x9d, representative alkyl analogues at position Y1 may be synthesized from the corresponding allyl as the starting material (such as eugenol for n-propyl) and involve hydrogenation of the allyl bond, followed by deprotection of the methyl ether using boron tribromide. Alternatively, as depicted by reaction xe2x80x9cbxe2x80x9d, the corresponding alkyl-phenol analogue may be employed as the starting material. followed by addition of the desired Z1 substituent. For example, alkyl phenol derivatives may be made by nitration using nitric acid, optionally followed by reduction with activated iron. In yet a further variation, the alkyl Z1 substituents can be introduced via Friedel Crafts alkylation of alkyl phenols via reaction xe2x80x9cbxe2x80x9d. Similarly, hydroxyalkylation of alkyl phenols can be achieved via treatment with an aldehyde and boron containing reagents, such as benzeneboronic acid. 
In addition, it should be recognized that the starting materials for the synthesis of compounds of structure (I) are commercially available from a number of sources.
In another aspect, the mitochondria protecting agents of this invention have the following structure (II): 
including steroisomers and pharmaceutically acceptable salts thereof, wherein
R1 and R2 are independently selected from xe2x80x94H, xe2x80x94C(xe2x95x90O)C1-3alkyl and C1-3alkyl;
X2 is optionally present and selected from xe2x80x94C(A2)(A3)xe2x80x94, xe2x80x94(CH2)nxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94NHxe2x80x94;
Y2 and Z2 are independently selected from xe2x80x94H, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NO2, xe2x80x94ORb, xe2x80x94NHCNHNH2, xe2x80x94NHRb and xe2x80x94NRbRc;
A2 and A3 are independently selected from xe2x80x94H and C1-3alkyl;
Rb and Rc are independently C1-4alkyl and
n is 2-9.
For purpose of clarity, position of Y2 and Z1 will be referenced by the following numbering: 
In one embodiment of structure (II), R1 and R2 are xe2x80x94H, Y2 and Z2 are xe2x80x94NH2, and the compounds of this invention has the following structure (II-1): 
In one embodiment of structure (II1), X2 is xe2x80x94C(A2)(A3)xe2x80x94, such as the following structure (II-2): 
In another embodiment of structure (II), R1 and R2 are xe2x80x94H, Y2 is xe2x80x94H and Z2 is xe2x80x94NH2, and the compounds of this invention has the following structure (II-3): 
Representative compounds of structure (II) are set forth in the following Table 2.
The compounds of structure (II) may be made by know organic reaction techniques, including those set forth in Example 7 below. For example, representative compounds of structure (II) may be made from the corresponding diphenol (such by 4,4-isopropylidenediphenol) as starting material as represented below by reaction xe2x80x9caxe2x80x9d. Nitration provides the mono- and di-nitro derivatives that can be separated by silica gel chromatography. Reduction of the nitro groups provide the corresponding amines. The amines can be further modified by reductive amination using paraformaldehyde and sodium cyanoborohydride. 4,4-isopropyldenediphenol can be reacted with methyl iodide in the presence of potassium carbonate to provide, for example, the monomethyl and dimethyl ethers via reaction xe2x80x9cbxe2x80x9d, which ethers may be separated by silica gel chromatography. These dialkyloxy derivatives may require more severe nitrating conditions, and nitroniurn tetrafluoroborate may be utilized to furnish the mono- and di-nitro systems as depicted by reaction xe2x80x9ccxe2x80x9d. Reduction of the nitro functionalities may be achieved using iron in aqueous acetic acid. 
In addition, it should be recognized that some of the compounds of structure (II), including starting materials therefor, are commercially available from a number of sources. Further, various references disclose additional techniques, such as published Japanese Application Nos. JP 07/278038 A2 to Hozumi et al. and JP 05/125180 A2 to Endo et al. (both of which are incorporated herein by reference), for the synthesis of compounds of structure (II).
In another aspect, the mitochondria protecting agents of this invention have the following structure (III): 
including steroisomers and pharmaceutically acceptable salts thereof, wherein
the dotted line represents a single or double bond;
A1 is selected from xe2x80x94H and C1-3alkyl;
Y3 is selected from xe2x80x94H, C1-3alkyl and xe2x80x94CORd;
Z3 is selected from xe2x80x94H, C1-3alkyl and xe2x80x94(CH2)mX3Rd;
each occurrence of X3 is selected from xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94NHxe2x80x94;
R3 is selected from xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2CH3 and xe2x80x94Rd;
Rd is selected from a guanidino moiety, a cylcoguanidino moiety, and a non-steroidal anti-inflammatory drug; and
m is 1-4.
In one embodiment of structure (III), R3 is Rd, and Rd is a guanidino moiety, and the compounds of this invention have the following structure (III-1): 
In another embodiment of structure (III), R3 is Rd, Rd is a cycloguanidino moiety, and the compounds of this invention have the following structure (III-2): 
In still a further embodiment of structure (III), Y3 is xe2x80x94CORd, where Rd is a non-steroidal anti-inflammatory drug (NSAID). Such NSAIDs may be coupled to the nitrogen at the Y3 position by formation of an amide linkage by, for example, reaction between a carboxylic acid moiety (xe2x80x94COOH) of the NSAID and secondary amine of structure (III) (xe2x80x94NHxe2x80x94). Thus, the designation xe2x80x9cxe2x80x94CORdxe2x80x9d should be understood to represent the formation of such an amide linkage. Representative compounds of this embodiment include those of the following structures (III7), (III-8) and (III-9): 
Representative compounds of structure (III) are set forth in the following Table 3.
The compounds of structure (III) may be made by know organic reaction techniques, including those set forth in Example 8 below. For example, representative compounds of structure (III) may be made from ethoxyquin or suitable 6-substituted 2,2,4-trimethyl, 1,2-dihydroquinolines as depicted below by reaction xe2x80x9caxe2x80x9d. Alkylation of the secondary amine may be carried out using reductive amination conditions. For example, acylation of the secondary amine to provide the NSAID derivatives may be accomplished by coupling with the corresponding acid halides at elevated temperatures. Alternatively, compounds of structure (III) may be made from the corresponding alcohol (or thiol) by reaction xe2x80x9cbxe2x80x9d, followed by conversion of the alcohol to the desired substituent. 
In addition, it should be recognized that some of the compounds of structure (III), including starting materials therefor, are commercially available from a number of sources. Further, various references disclose additional techniques, such as published Japanese Application No. JP 63/058455 A2 to Tamaki et al. and German Patent No. DE 2156371 (both of which are incorporated herein by reference), for the synthesis of compounds of structure (III).
In another aspect, the mitochondria protecting agents of this invention have the following structure (IV): 
including steroisomers and pharmaceutically acceptable salts thereof, wherein
W1, W2 and W3 are independently selected from xe2x80x94H and C1-3alkyl;
X4 is optionally selected from xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 and xe2x80x94Sxe2x80x94;
Y4 is selected from xe2x80x94H and C1-12alkyl; and
R4 is selected from xe2x80x94H, a guanidino moiety, a cylcoguanidino moiety, and a non-steroidal anti-inflammatory drug, or X4xe2x80x94R4 taken together is selected from xe2x80x94CH2OH, xe2x80x94OC(xe2x95x90O)CH3, and C1-3 alkyl.
In one embodiment of structure (IV), R4 is a guanidino moiety, and the compounds of this invention have the following structure (IV-1): 
In another embodiment of structure (IV), R4 is cycloguanidino moiety, and the compounds of this invention have the following structure (IV-2): 
In another embodiment of structure (IV), X4 is O and R4 is H as represented by structure (IV-3), or X4 and R4 taken together is a C1-3alkyl as represented by structure (IV-4): 
In still a further embodiment of structure (IV), R4 is a non-steroidal anti-inflammatory drug (NSAID). Such NSAIDs may be coupled via X4 by formation of a suitable bond, such as an amide, ester or thioester linkage. Thus, when X4 is oxygen, a an amide or ester may be formed to join the NSAID. For example, an ester linkage may be formed to join a compound of structure (IV) to naproxen, aspirin and ibuprofen as represented by the following structures (IV-6), (IV-7) and (IV-8): 
Representative compounds of structure (IV) are set forth in the following Table 4.
The compounds of structure (IV) may be made by know organic reaction techniques, including those set forth in Example 9 below. For example, representative compounds of structure (IV) may be made from 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as depicted below by reaction xe2x80x9caxe2x80x9d. Reduction of the acid group to the alcohol followed by optionally by coupling with a variety of different moieties, such as an NSAID, provides the corresponding compounds of structure (IV). For example, an aminoguanidine derivative may be prepared by reductive amination of the aldehyde using aminoguanidine and sodium cyanoborohydride. The aldehyde can be formed by Swem oxidation of the primary alcohol as described in the synthesis of Example 9 (see synthetic procedure of compound (IV-9)). The chroman system containing a methylamine substituent may be prepared by hydride reduction of the amide of Trolox via reaction xe2x80x9cbxe2x80x9d. Keeping the phenolic hydroxyl protected, the amine may be coupled to a variety of moieties, such as NSAIDs, via reaction xe2x80x9ccxe2x80x9d. Deprotection of the hydroxyl group then provides compounds of structure (IV) wherein xe2x80x94X4xe2x80x94R4 is, for example, xe2x80x94NHxe2x80x94NSAID. 
In addition, it should be recognized that the starting materials for the synthesis of compounds of structure (IV) are commercially available from a number of sources.
Pharmaceutically acceptable salts of the compounds of this invention may be made by techniques well known in the art, such as by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water of in an organic solvent. Suitable salts in this context may be found in Remington""s Pharmaceuitcal Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985, which is hereby incorporated by reference.
A mitochondria protecting agent of this invention, or a pharmaceutically acceptable salt thereof, is administered to a patient in a therapeutically effective amount. A therapeutically effective amount is an amount calculated to achieve the desired effect. It will be apparent to one skilled in the art that the route of administration may vary with the particular treatment. Routes of administration may be either non-invasive or invasive. Non-invasive routes of administration include oral, buccal/sublingual, rectal, nasal, topical (including transdermal and ophthalmic), vaginal, intravesical, and pulmonary. Invasive routes of administration include intarterial, intravenous, intradermal, intramuscular, subcutaneous, intraperitoneal, intrathecal and intraocular.
The required dosage may vary with the particular treatment and route of administration. In general, dosages for mitochondria protecting agents will be from about 1 to about 5 milligrams of the compound per kilogram of the body weight of the host animal per day; frequently it will be between about 100 xcexcg and about 5 mg but may vary up to about 50 mg of compound per kg of body weight per day. Therapeutic administration is generally performed under the guidance of a physician, and pharmaceutical compositions contain the mitochondria protecting agent in a pharmaceutically acceptable carrier. These carriers are well known in the art and typically contain non-toxic salts and buffers. Such carriers may comprise buffers like physiologically-buffered saline. phosphate-buffered saline, carbohydrates such as glucose, mannose, sucrose, mannitol or dextrans, amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants and preservatives. Acceptable nontoxic salts include acid addition salts or metal complexes, eg., with zinc, iron, calcium, barium, magnesium, aluminum or the like (which are considered as addition salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, tannate. oxalate, famarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
Mitochondria protecting agents of this invention also include prodrugs thereof. As used herein, a xe2x80x9cprodrugxe2x80x9d is any covalently bonded carrier that releases in vivo the active parent drug according the structures (I) through (IV) when such prodrug is administered to the animal. Prodrugs of the compounds of structures (I) through (IV) are prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include, but are not limited to, compounds of structures (I) through (IV) wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to the animal, cleaves to form the free hydroxyl, amino or sulfhydryl group, respectively. Representative examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups.
The effectiveness of a compound of this invention as a mitochondria protecting agent may be determined by various assay methods. Suitable mitochondria protecting agents are active in one or more of the following assays for maintenance of mitochondrial structural and functional integrity, or in any other assay known in the art that measures the maintenance of mitochondrial structural and functional integrity. Accordingly, it is an aspect of the invention to provide methods for treating mitochondria associated diseases that include methods of administering compounds that may or may not have known antioxidant properties. However, according to this aspect of the invention, the unexpected finding is disclosed herein that mitochondria protecting agents may exhibit mitochondria protecting activities that are not predictable based upon determination of antioxidant properties in non-mitochondrial assay systems.
According to this assay, the ability of a mitochondria protecting agent of the invention to inhibit production of ROS intracellularly may be compared to its antioxidant activity in a cell-free environment. Production of ROS may be monitored using, for example by way of illustration and not limitation, 2xe2x80x2,7xe2x80x2-dichlorodihydroflurescein diacetate (xe2x80x9cdichlorofluorescin diacetatexe2x80x9d or DCFC), a sensitive indicator of the presence of oxidizing species. Non-fluorescent DCFC is converted upon oxidation to a fluorophore that can be quantified fluorimetrically. Cell membranes are also permeable to DCFC, but the charged acetate groups of DCFC are removed by intracellular esterase activity, rendering the indicator less able to diffuse back out of the cell.
In the cell-based aspect of the DCFC assay for inhibition of production of ROS, cultured cells may be pre-loaded with a suitable amount of DCFC and then contacted with a mitochondria protecting agent. After an appropriate interval, free radical production in the cultured cells may be induced by contacting them with iron (III)/ascorbate and the relative mean DCFC fluorescence can be monitored as a function of time.
In the cell-free aspect of the DCFC assay for inhibition of production of ROS, a mitochondria protecting agent may be tested for its ability to directly inhibit iron/ascorbate induced oxidation of DCFC when the protecting agent, the fluorescent indicator and the free radical former are all present in solution in the absence of cells.
Comparison of the properties of a mitochondria protecting agent in the cell-based and the cell-free aspects of the DCFC assay may permit determination of whether inhibition of ROS production by a mitochondria protecting agent proceeds stoichiometrically or catalytically. Without wishing to be bound by theory, mitochondria protecting agents that scavenge free radicals stoichiometrically (e.g., on a one-to-one molecular basis) may not represent preferred agents because high intracellular concentrations of such agents might be required for them to be effective in vivo. On the other hand, mitochondria protecting agents that act catalytically may moderate production of oxygen radicals at their source. or may block ROS production without the agents themselves being altered, or may alter the reactivity of ROS by an unknown mechanism. Such mitochondria protecting agents may xe2x80x9crecyclexe2x80x9d sothat they can inhibit ROS at substoichiometric concentrations. Determination of this type of catalytic inhibition of ROS production by a mitochondria protecting agent in cells may indicate interaction of the agent with one or more cellular components that synergize with the agent to reduce or prevent ROS generation. A mitochondria protecting agent having such catalytic inhibitor; characteristics may be a preferred agent for use according to the method of the invention.
Mitochondria protecting agents that are useful according to the instant invention may inhibit ROS production as quantified by this fluorescence assay or by other assays based on similar principles. A person having ordinary skill in the art is familiar with variations and modifications that may be made to the assay as described here without departing from the essence of this method for determining the effectiveness of a mitochondria protecting agent, and such variations and modifications are within the scope of this disclosure.
According to this assay, one may determine the ability of a mitochondria protecting agent of the invention to inhibit the loss of mitochondrial membrane potential that accompanies mitochondrial dysfunction. As noted above, maintenance of a mitochondrial membrane potential may be compromised as a consequence of mitochondrial dysfunction. This loss of membrane potential or mitochondrial permeability transition (MPT) can be quantitatively measured using the mitochondria-selective fluorescent probe 2-,4-dimethylarninostyryl-N-methylpyridinium (DASPMI).
Upon introduction into cell cultures, DASPMI accumulates in mitochondria in a manner that is dependent on, and proportional to, mitochondrial membrane potential. If mitochondrial function is disrupted in such a manner as to compromise membrane potential, the fluorescent indicator compound leaks out of the membrane bounded organelle with a concomitant loss of detectable fluorescence. Fluorimetric measurement of the rate of decay of mitochondria associated DASPMI fluorescence provides a quantitative measure of loss of membrane potential, or MPT. Because mitochondrial dysfunction may be the result of reactive free radicals such as ROS, mitochondria protecting agents that retard the rate of loss of DASPMI fluorescence may be effective agents for treating mitochondria associated diseases according to the methods of the instant invention.
As noted above, mitochondrial dysfunction may be an induction signal for cellular apoptosis. According to this assay, one may determine the ability of a mitochondria protecting agent of the invention to inhibit or delay the onset of apoptosis. Mitochondrial dysfunction may be present in cells known or suspected of being derived from a subject with a mitochondria associated disease, or mitochondrial dysfunction may be induced in normal cells by one or more of a variety of physiological and biochemical stimuli with which those having skill in the art will be familiar.
In one aspect of the apoptosis assay, translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is quantified by measuring outer leaflet binding by the PS-specific protein annexin. (Martin et al, J. Exp. Med. 182:1545, 1995; Fakok et al., J. Immunol. 148:2207, 1992.) In another aspect of the apoptosis assay, induction of specific protease activity in a family of apoptosis-activated proteases known as the caspases is measured, for example by determination of caspase-mediated cleavage of specifically recognized protein substrates. These substrates may include, for example, poly-(ADP-ribose) polymerase (PARP) or other naturally occurring or synthetic peptides and proteins cleaved by caspases that may be known in the art (See, e.g, Ellerby et al., J. Neurosci. 17:6165-6178, 1997.) In another aspect of the apoptosis assay, quantification of the mitochondrial protein cytochrome c that has leaked out of mitochondria in apoptotic cells may provide an apoptosis indicator that can be readily determined. (Liu et al., Cell 86:147, 1996) Such quantification of cytochrome c may be performed spectrophotometrically, immunochemically or by other well established methods for detecting the presence of a specific protein. A person of ordinary skill in the art will readily appreciate that there may be other suitable techniques for quantifying apoptosis, and such techniques for purposes of determining the effects of mitochondria protecting agents on the induction and kinetics of apoptosis are within the scope of the assays disclosed here.
As described above, mitochondria associated diseases may be characterized by impaired mitochondrial respiratory activity that may be the direct or indirect consequence of elevated levels of reactive free radicals such as ROS. Accordingly, a mitochondria protecting agent for use in the methods provided by the instant invention may restore or prevent further deterioration of ETC activity in mitochondria of individuals having mitochondria associated diseases. Assay methods for monitoring the enzymatic activities of mitochondrial ETC Complexes I, II, III, IV, and ATP synthetase, and for monitoring oxygen consumption by mitochondria, are well known in the art. (See, e.g., Parker et al., Neurology 44:1090-96, 1994; Miller et al, J. Neurochem. 67:1897, 1996.) It is within the scope of the methods provided by the instant invention to identify a mitochondria protecting agent using such assays of mitochondrial function. Further, mitochondrial function may be monitored by measuring the oxidation state of mitochondrial cytochrome c at 540 nm. As described above, oxidative damage that may arise in mitochondria associated diseases may include damage to mitochondrial components such that cytochrome c oxidation state, by itself or in concert with other parameters of mitochondrial function including but not limited to mitochondrial oxygen consumption, may be an indicator of reactive free radical damage to mitochondrial components. Accordingly, the invention provides various assays designed to test the inhibition of such oxidative damage by mitochondria protecting agents. The various forms such assays may take will be appreciated by those familiar with the art and is not intended to be limited by the disclosures herein, including in the Examples.
For example by way of illustration and not limitation, Complex IV activity may be determined using commercially available cytochrome c that is fully reduced via exposure to excess ascorbate. Cytochrome c oxidation may then be monitored spectrophotometrically at 540 nm using a stirred cuvette in which the ambient oxygen above the buffer is replaced with argon. Oxygen reduction in the cuvette may be concurrently monitored using a micro oxygen electrode with which those skilled in the art will be familiar, where such an electrode may be inserted into the cuvette in a manner that preserves the argon atmosphere of the sample, for example through a sealed rubber stopper. The reaction may be initiated by addition of a cell homogenate or, preferably a preparation of isolated mitochondria, via injection through the rubber stopper. This assay, or others based on similar principles, may permit correlation of mitochondrial respiratory activity with structural features of one or more mitochondrial components. In the assay described here, for example, a defect in Complex IV activity may be correlated with an enzyme recognition site.